DsrA protein and polynucleotides encoding the same

ABSTRACT

DsrA is an outer membrane protein of  H. ducreyi  that confers serum resistance to the bacteria. Isolated polynucleotides encoding the protein, end expression vectors and host cells encoding the same, are described. Also described is a mutant  H. ducreyi  strain that does not express DsrA. Vaccines against  H. ducreyi  and methods of using the same are also described.

This application claims priority to PCT Application numberPCT/US00/18834 filed in English on Jul. 7, 2000 claming priority fromU.S. Provisional Patent Application No. 60/143,257 filed on Jul. 9,1999, the disclosures of which are hereby incorporated herein byreference in their entirety.

STATEMENT OF FEDERAL SUPPORT

This invention was made with United States Government support undergrant numbers Al 40263 and A126837 from the National Institutes ofHealth (Public Health Service). The United States Government has certainrights to this invention.

FIELD OF THE INVENTION

This invention relates to proteins that are involved in the serumresistance of H. ducreyi.

BACKGROUND OF THE INVENTION

Haemophilus ducreyi is the etiologic agent of chancroid, a genital ulcerdisease transmitted by sexual contact. See, e.g. Albritton, W. L.,Microbiol Rev. 53:377–89 (1989); Trees, D. L., and S. A. Morse, ClinMicrobiol Rev. 8, 357–375 (1995). Chancroid has gained importancerecently because it has been implicated as an independent risk factorfor the heterosexual transmission of HIV in Africa. See Albritton, supraTrees, supra: R. M. Greenblattet et al., AIDS 2, 47–50 (1988);Jessamine, P. G., and A. R. Ronald, Med Clin North Am. 74, 1417–31(1990); Plummer, F. A. et al., J Infect Dis. 161, 810–1 (1990); D. L.,and S. A. Morse, Clin. Microbiol Rev. 8, 357–375 (1995): Wasserheit, J.N., Sex Trans Dis. 19, 61–77 (1991).

Serum resistance has been shown in numerous bacterial systems to becritical for the survival of invading bacterial and the establishment ofdisease, since mutations which result in the loss of serum resistancerenders several bacterial pathogens avirulent. See. e.g., Blaser, M. J.,American Journal of the Medical Sciences. 306, 325–9 (1993); Corbeil, L.B., Canadian Journal of Veterinary Research. 54,S57–62 (1990), Mobley,H. L. et al., Kidney International—Supplement. 47, S129–36 (1994); Rice,P. A., Clinical Microbiology Review. 2, S112–7 (1989); and Stull, T. L.,and J. J. LiPuma, Medical Clinics of North America. 75, 287–9 (1991). Inmost systems, the serum resistance phenotype is the product of multiplegenes. H. ducreyi is resistant to high levels of normal human serum(NHS; up to 50%). Early studies on H. ducreyi serum resistance byOdumeru and colleagues concluded that truncation of LOS in severalstrains was associated with avirulence and loss of serum resistance (seeOdumeru, J. A. et al., Infect. Immun. 43, 607–611 (1984); Odumeru, J. A.et al., Infect. Immun. 50, 495–9 (1985); Odumeru, J. A. et al., J MedMicrobiol. 23, 155–62 (1987)), whereas a recent study came to theopposite conclusion. See Hiltke, T. J. et al., Microb Path. 26,93–102(1999)

Originally described as a cell spreading factor, vitronectin is nowrecognized as a multifunctional regulatory adhesive glycoproteininvolved in a variety of extracellular processes such as the attachmentand spreading of normal and neoplastic cells, as well as the function ofthe complement and coagulation pathways. Integrins are transmembrane αβheterodimer receptors expressed on a wide variety of cells which areinvolved in extracellular matrix interactions. The ligands for severalof the integrins are adhesive extracellular matrix (ECM) proteins suchas fibronectin, vitronectin, collagens and laminin.

Proteins or fragments thereof that are able to interfere withvitronectin binding to various integrins and to block integrin-mediatedcell attachment to extracellular matrix proteins are useful inpreventing the attachment of the bacteria to the host organism, and thusinfection of the host.

The ability to use a protein or antibody that interferes withvitronectin binding in a vaccine against H. ducreyi is desirable. Thesekinds of proteins are believed to be highly conserved among strains of aparticular type of bacteria in that they are the protein molecules thatmediate attachment by bonding bacteria to host cells, the initial stepin the infection process. A vaccine against H. ducreyi comprising aprotein or antibody that would interfere with vitronectin binding wouldbe effective against a broad array of types and strains of H. ducreyi.The use of such a vaccine may prevent adherence of the bacteria to thetissue of the host animal. In that adherence is one of the initial stepin H. ducreyi infection, accordingly, preventing or limiting theinfection at this point would be advantageous.

In view of the foregoing, it would be desirable to determine themechanism of serum resistance in H. ducreyi. Additionally, thedevelopment of an effective vaccine against H. ducreyi would beadvantageous.

SUMMARY OF THE INVENTION

Certain objects, advantages and novel features of the invention will beset forth in the description that follows, and will become apparent tothose skilled in the art upon examination of the following, or may belearned with the practice of the invention.

The present invention is based in the inventor's discovery that aprotein, referred to herein as DsrA (Ducreyi Serum Resistance Aprotein), has been found to play a critical role in the resistance of H.ducreyi to normal human serum.

Accordingly, one aspect of the invention is a polynucleotide (e.g., DNA)that encodes the protein DsrA. Particularly preferred is the DNA of SEQID NO:1, which encodes the protein DsrA set forth in SEQ ID NO:2.

An additional aspect of the invention is the isolated protein DsrA,which protein may vary in molecular weight between 28 and 35kilodaltons, depending on whether the particular DsrA protein sequencecomprises one, two or three copies of the amino acid heptamer NTHNINK(SEQ ID NO:19).

Expression vectors and host cells expressing DsrA are also an aspect ofthe invention. Antibodies against DsrA and antisense molecules of DsrAare a further aspect of the present invention.

Vaccines against H. ducreyi comprising proteins, polynucleotides andexpression vectors of DsrA are a further aspect of the invention.

Also an aspect of this invention is an isogenic mutant (FX517) of H.ducreyi strain 35000 that does not express DsrA, which mutant finds usein an attenuated vaccine against H. ducreyi.

The foregoing and other aspects of the present invention are explainedin detail in the specification set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph of Western Blot illustrating the distribution ofthe DsrA protein and summary of serum resistance of H. ducreyi strains.Total cellular proteins from geographically diverse H. ducreyi strainswere subjected to SDS-PAGE and Western blotting using anti-DsrA mousesera. Bound antibody was detected with alkaline phosphatase-conjugatedsecondary antibody and BCIP/NBT substrate. An additional twelve H.ducreyi strains also expressed a 28–35 kDa protein which reacted withthis serum (data not shown). The names of strains are indicated aboveeach lane. Shown to the left of the gel are molecular weight standards,where the abbreviation kDa means kilodaltons. R refers to resistant to50% NHS; S, sensitive to 50% NHS, an asterisk indicates that resistanceto NHS was indeterminate. The data in FIG. 1 are compiled fromexperiments done on at least three separate days.

FIG. 2 is a schematic illustration of the restriction map of the dsrAregion and PCR products thereof. The dsrA open reading frame is boxed.The restriction sites are indicated. The numbered arrows indicatedirection and position of the dsrA oligos used for PCR. The letter KSand T7 (promoter) refer to the vector primers used in thevector-anchored PCR reactions. V-A PCR refers to vector-anchored PCR; Prefers to a promoter. The jagged lines represent approximately 2 kb ofsequence not shown downstream of the dsrA locus.

FIG. 3 sets forth the DNA sequence (SEQ ID NO:1) and deduced amino acidsequence (SEQ ID NO:2) of the dyrA locus. The putative −35 and −10promoter sequences are indicated and underlined. A putative ribosomebinding site is labeled RBS and underlined. Twenty one amino acidscomprising the signal peptide are underlined. The stop codon TAA isindicated with an asterisk. The opposing arrows show a potential stemloop transcription terminator.

FIG. 4 sets forth a comparison of the amino acid sequence of DsrA (SEQID NO:2) with the UspA2 protein of M. catarrahalis (SEQ ID NO:20) andthe YadA protein of Y. enterocolitica (SEQ ID NO:21). Shaded, boxedresidues indicate homologous sequences.

FIGS. 5A and 5B show a SDS-PAGE/Western blot of parent strain 35000 anddsrA mutant FX517. Outer membranes were prepared, solubilized at 37° C.or 100° C. and subjected to SDS-PAGE and Coomassie staining (Panel A).For the Western blot (panel B), outer membranes were solubilized at 100°C., transferred to nitrocellulose and probed with anti-DsrA mouse serum.Bound antibody was detected with alkaline phosphatase-conjugatedsecondary antibody and BCIP/NBT substrate. The asterices indicate thepositions of the DsrA protein. STD, molecular weight standards.

FIG. 6 is a graphical illustration of the bactericidal killing of parentstrain 35000 compared with the bactericidal killing of the dsrA mutantFX517. Bactericidal killing was performed as described in FIG. 1, exceptthat two serum concentrations were utilized. The data presented in FIGS.1 and 6 for 35000 with 50% sera are the same data. The data presentedfor 35000 were obtained in parallel experiments with FX517.

FIG. 7 is a photograph of a SDS-PAGE/Western Blot illustratingComplementation of dsrA mutants. Total cellular proteins from theindicated H. ducreyi strains were subjected to SDS-PAGE (12%) andWestern blotting using anti-DsrA antisera. Bound antibody was detectedwith horseradish peroxidase-conjugated secondary antibody followed bychemiluminescence. “N” indicates no plasmid present; “+” indicates pUNCH1260 (i.e., contains the entire dyrA ORF from strain 35000 and itsputative native promoter as illustrated in FIG. 2); “−” indicates pLSKSa vector without insert. Below each strain are shown the summary ofbactericidal killing of the complemented dsrA mutants. Bactericidalkilling was performed as in FIG. 1 (50% serum), except that the mediumused contained streptomycin.

FIG. 8 is a photograph of an SDS-PAGE gel illustrating the analysis ofLOS as described in Example 16, below. Crude LOS was prepared asdescribed in the text and subjected to SDS-PAGE and silver staining.

FIG. 9 illustrates a comparison of the deduced amino acid sequences ofdsrA from strain 35000 (SEQ ID NO:2) and eight additional H. ducreyistrains (CIP A75. SEQ ID NO:4. CIP A77. SEQ ID NO:6; CIP542 (CAN). SEQID NO:8; CIP542 (CDC), SEQ ID NO:10; CHIA, SEQ ID NO:12 V-1157. SEQ IDNO:14: M90-02. SEQ ID NO:16 and 406, SEQ ID NO:18). Variable regions 1and 2 are indicated.

FIG. 10 illustrates the promoter regions of dsrA from various strains ofH. ducreyi (35000, CIP542 (CAN), CIP542 (CDC). CHIA, V-1157, M90-02 and406, SEQ ID NO:22, CIP A75 and CIP A77, SEQ ID NO:23) and the mutationsin the strains CIP A75 and CIP A77, which do not express DsrA. The 5base-pair deletions present in strains CIP A75 and CIP A77 are shown ashyphens.

FIG. 11 is a graphical illustration showing that efficient attachment ofH. ducreyi to a keratinocyte cell line requires DsrA expression. H.ducreyi were added to HaCaT cells at a MOI of between 1–5:1 andincubated for two hours. After removal of unbound bacteria by extensivewashing, CFUs were determined by plating the disrupted monolayer. Thedata shown in FIG. 11 are taken from four experiments.

FIG. 12 is an autoradiograph of an SDS-PAGE illustrating the affinitypurification of DsrA from whole cells using biotinylated vitronectins(Vn). Biotinylated vitronectins were mixed with surface-iodinated H.ducreyi and allowed to bind. After washing unbound vitronectin bycentrifugation and washing, H. ducreyi were solubilized with a gentledetergent. Total soluble H. ducreyi proteins were bound to solid-phasestreptavidin-agarose. After washing the streptavidin agarose, boundproteins were eluted by boiling in sample buffer and analysis bySDS-PAGE and autoradiography.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention will now be described more fully hereinafter withreference to the accompanying figures, in which preferred embodiments ofthe invention are shown. This invention may, however, be embodied indifferent forms and should not be construed as limited to theembodiments set forth herein. Rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the invention to those skilled in the art.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety.

Amino acid sequences disclosed herein are presented in the amino tocarboxy direction, from left to right. The amino and carboxy groups arenot presented in the sequence. Nucleotide sequences are presented hereinby single strand only, in the 5′ to 3′ direction, from left to right.Nucleotides and amino acids are represented herein in the mannerrecommended by the IUPAC-IUB Biochemical Nomenclature Commission, or(for amino acids) by three letter code, in accordance with 37 CFR §1.822 and established usage. See, e.g., Patent In User Manual, 99–102(November 1990) (U.S. Patent and Trademark Office).

DsrA is an H. ducreyi outer membrane protein required for the expressionof serum resistance and is encoded by the gene dsrA, described herein.The isolated H. ducreyi protein DsrA, and the isolated polynucleotidesthat encode the protein, are aspects of the present invention. The DsrAprotein in its monomer form varies in molecular weight between 28 and 35kDA between different H. ducreyi strains in SDS-PAGE and Western blots.The dsrA locus from several H. ducreyi strains was sequenced and thededuced amino acid sequences were greater than 85% identical. The majordifference between the different strains is found in the amino acidsequence, in which either one, two or three copies of the amino acidsequence NTHNINK (SEQ ID NO:19) are present in the VR2 region of theprotein; these repeats account for the variability in the monomer formof the DsrA observed in SDS-PAGE. DsrA proteins that contain one, two orthree copies of the NTHNINK (SEQ ID NO:19) in the VR2 region of theprotein, and accordingly having a molecular weight of between 28 and 35kilodaltons, are all within the scope of the present invention.Additionally, DsrA, as used herein, refers to the amino acid sequencesof substantially purified DsrA obtained from any species, particularlymammalian, including bovine, ovine, porcine, murine, equine, andpreferably human, from any source whether natural, synthetic,semi-synthetic, or recombinant.

As used herein, in this context, the term “isolated” means that theprotein is significantly free of other proteins. That is, a compositioncomprising the isolated protein is between 70% and 94% pure by weight.Preferably, the protein is purified. As used herein, the term “purified”and related terms means that the protein is at least 95% pure by weight,preferably at least 98% pure by weight, and most preferably at least 99%pure by weight.

An “allele” as used herein, is an alternative form of thepolynucleotides (i.e., genes) encoding DsrA. Alleles may result from atleast one mutation in the nucleic acid sequence and may result inaltered mRNAs or polypeptides whose structure or function may or may notbe altered. Any given natural or recombinant gene may have none, one, ormany allelic forms. Common mutational changes which give rise to allelesare generally ascribed to natural deletions, additions, or substitutionsof nucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

“Amino acid sequence,” as used herein, refers to an oligopeptide,peptide, polypeptide, or protein sequence, and fragment thereof, and tonaturally occurring or synthetic molecules. Fragments of DsrA arepreferably and retain the biological activity or the immunologicalactivity of DsrA. Where “amino acid sequence” is recited herein to referto an amino acid sequence of a naturally occurring protein molecule,amino acid sequence, and like terms, are not meant to limit the aminoacid sequence to the complete, native amino acid sequence associatedwith the recited protein molecule.

“Amplification”, as used herein, refers to the production of additionalcopies of a nucleic acid sequence and is generally carried out usingpolymerase chain reaction (PCR) technologies well known in the art(Dieffenbach, C. W. and G. S. Dveksler. PCR Primer, a Laboratory Manual,Cold Spring Harbor Press, Plainview, N.Y.(1995)).

As used herein, the term “antibody” refers to intact molecules as wellas fragments thereof, such as Fa, F(ab′)2, and Fc, which are capable ofbinding the DsrA protein or an antigenic or epitopic determinantthereof. Antibodies that bind DsrA polypeptides can be prepared usingintact polypeptides or fragments containing small peptides of interestas an immunizing antigen. The polypeptide or oligopeptide may be used toimmunize an animal and can be derived from the translation of RNA orsynthesized chemically and can be conjugated to a carrier protein, ifdesired. Commonly used carriers that are chemically coupled to peptidesinclude bovine serum albumin and thyroglobulin, keyhole limpethemocyanin. The coupled peptide is then used to immunize the animal(e.g., a mouse, a rat, or a rabbit).

The term “antigenic determinant”, as used herein, refers to thatfragment of a molecule (i.e. an epitope) that makes contact with aparticular antibody. When a protein or fragment of a protein is used toimmunize a host animal, numerous regions of the protein may induce theproduction of antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

The term “antisense”, as used herein, refers to any compositioncontaining nucleotide sequences which are complementary to a specificDNA or RNA sequence. The term “antisense strand” is used in reference toa nucleic acid strand that is complementary to the “sense” strand.Antisense molecules include peptide nucleic acids and may be produced byany method including synthesis or transcription. Once introduced into acell, the complementary nucleotides combine with natural sequencesproduced by the cell to form duplexes and block either transcription ortranslation. The designation “negative” is sometimes used in referenceto the antisense strand, and “positive” is sometimes used in referenceto the sense strand.

The terms “complementary” or “complementarity,” as used herein, refer tothe natural binding of polynucleotides under permissive salt andtemperature conditions by base-pairing. For example, the sequence“A-G-T” binds to the complementary sequence “T-C-A”. Complementaritybetween two single-stranded molecules may be “partial,” in which onlysome of the nucleic acids bind, or it may be complete when totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of hybridization between nucleic acidstrands.

A “deletion”, as used herein, refers to a change in the amino acid ornucleotide sequence and results in the absence of one or more amino acidresidues or nucleotides.

The term “derivative”, as used herein, refers to the chemicalmodification of a nucleic acid encoding or complementary to DsrA or theencoded DsrA. Such modifications include, for example, replacement ofhydrogen by an alkyl, acyl, or amino group. A nucleic acid derivativeencodes a polypeptide which retains the biological or immunologicalfunction of the natural molecule. A derivative polypeptide is one whichis modified by glycosylation, pegylation, or any similar process whichretains the biological or immunological function of the polypeptide fromwhich it was derived.

The term “homology”, as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence that at leastpartially inhibits an identical sequence from hybridizing to a targetnucleic acid is referred to using the functional term “substantiallyhomologous.” The inhibition of hybridization of the completelycomplementary sequence to the target sequence may be examined using ahybridization assay (Southern or northern blot, solution hybridizationand the like) under conditions of low stringency. A substantiallyhomologous sequence or hybridization probe will compete for and inhibitthe binding of a completely homologous sequence to the target sequenceunder conditions of low stringency. This is not to say that conditionsof low stringency are such that non-specific binding is permitted; lowstringency conditions require that the binding of two sequences to oneanother be a specific (i.e., selective) interaction. The absence ofnon-specific binding may be tested by the use of a second targetsequence which lacks even a partial degree of complementarity (e.g.,less than about 30% identity). In the absence of non-specific binding,the probe will not hybridize to the second non-complementary targetsequence.

The term “hybridization”, as used herein, refers to any process by whicha strand of nucleic acid binds with a complementary strand through basepairing. The term “hybridization complex”, as used herein, refers to acomplex formed between two nucleic acid sequences by virtue of theformation of hydrogen bonds between complementary G and C bases andbetween complementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g. C₀t or R₀tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,paper, membranes, filters, chips, pins or glass slides, or any otherappropriate substrate to which cells or their nucleic acids have beenfixed).

An “insertion” or “addition”, as used herein, refers to a change in anamino acid or nucleotide sequence resulting in the addition of one ormore amino acid residues or nucleotides, respectively, as compared tothe naturally occurring molecule.

“Nucleic acid sequence” as used herein refers to an oligonucleotide,nucleotide, or polynucleotide, and fragments thereof, and to DNA or RNAof genomic or synthetic origin which may be single- or double-stranded,and represent the sense or antisense strand. “Fragments” are thosenucleic acid sequences which are greater than 60 nucleotides than inlength, and most preferably includes fragments that are at least 100nucleotides or at least 1000 nucleotides, and at least 10,000nucleotides in length.

The term “oligonucleotide” refers to a nucleic acid sequence of at leastabout 6 nucleotides to about 60 nucleotides, preferably about 15 to 30nucleotides, and more preferably about 20 to 25 nucleotides, which canbe used in PCR amplification or a hybridization assay, or a microarray.As used herein, oligonucleotide is substantially equivalent to the terms“amplimers”, “primers”, “oligomers”, and “probes”, as commonly definedin the art.

The term “sample”, as used herein, is used in its broadest sense. Abiological sample suspected of containing nucleic acid encoding DsrA, orfragments thereof, or DsrA itself may comprise a bodily fluid, extractfrom a cell, chromosome, organelle, or membrane isolated from a cell, acell, genomic DNA, RNA, or cDNA (in solution or bound to a solidsupport, a tissue, a tissue print, and the like).

The terms “stringent conditions” or “stringency”, as used herein, referto the conditions for hybridization as defined by the nucleic acid,salt, and temperature. These conditions are well known in the art andmay be altered in order to identify or detect identical or relatedpolynucleotide sequences. Numerous equivalent conditions comprisingeither low or high stringency depend on factors such as the length andnature of the sequence (DNA, RNA, base composition), nature of thetarget (DNA, RNA, base composition), milieu (in solution or immobilizedon a solid substrate), concentration of salts and other components(e.g., formamide, dextran sulfate and/or polyethylene glycol), andtemperature of the reactions (within a range from about 5° C. below themelting temperature of the probe to about 20° C. to 25° C. below themelting temperature). One or more factors may be varied to generateconditions of either low or high stringency different from, butequivalent to, the above listed conditions.

A “substitution”, as used herein, refers to the replacement of one ormore amino acids or nucleotides by different amino acids or nucleotides,respectively.

“Transformation”, as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the type of host cell beingtransformed and may include, but is not limited to, viral infection,electroporation, heat shock, lipofection, and particle bombardment. Such“transformed” cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

Polynucleotides of the present invention include those polynucleotidesencoding for proteins homologous to, and having essentially the samebiological properties as, the protein DsrA disclosed herein.Particularly preferred is the DNA disclosed herein as SEQ ID NO:1 andencoding the protein DsrA given herein SEQ ID NO:2. This definition ofpolynucleotides of the present invention is intended to encompassnatural allelic sequences thereof. Accordingly, other preferredembodiments of the present invention include the polynucleotides setforth herein as SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQID NO:11, SEQ ID NO:13, SEQ ID NO:15, and SEQ ID NO:17, whichpolynucleotide sequences encode the protein sequences set forth as SEQID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, and SEQ ID NO:18, respectively. Isolated DNA orcloned genes of the present invention can be of any species of origin,including mouse, rat, rabbit, cat, porcine, and human, but arepreferably of mammalian origin. Polynucleotides that hybridize to anyone of the DNA disclosed herein as SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ II)NO:15, or SEQ ID NO:17 (or fragments or derivatives thereof which serveas hybridization probes as discussed below) and which code on expressionfor a protein of the present invention (e.g. a protein according to SEQID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18) are also an aspectof the invention. Conditions which will permit other polynucleotidesthat code on expression for a protein of the present invention tohybridize to the any one of DNA of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ IDNO:15, or SEQ ID NO:17 disclosed herein can be determined in accordancewith known techniques. For example, hybridization of such sequences maybe carried out under conditions of reduced stringency, medium stringencyor even stringent conditions (e.g. conditions represented by a washstringency of 35–40% Formamide with 5× Denhardt's solution, 0.5% SDS and1× SSPE at 37° C.; conditions represented by a wash stringency of 40–45%Formamide with 5× Denhardt's solution, 0.5% SDS, and 1× SSPE at 42° C.;and conditions represented by a wash stringency of 50% Formamide with 5×Denhardt's solution, 0.5% SDS and 1× SSPE at 42° C., respectively) toany one of the DNA of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, or SEQ IDNO:17 disclosed herein in a standard hybridization assay. See, e.g., J.Sambrook et al., Molecular Cloning, A Laboratory Manual (2d Ed. 1989)(Cold Spring Harbor Laboratory). In general, sequences which code forproteins of the present invention and which hybridize to any one of theDNA of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9,SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, or SEQ ID NO:17 disclosedherein will be at least 75% homologous, 85% homologous, and even 95%homologous or more with the any one of SEQ ID NO:1. SEQ ID NO:3. SEQ IDNO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ IDNO:15, or SEQ ID NO:17. Further, polynucleotides that code for proteinsof the present invention, or polynucleotides that hybridize to any oneof SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQID NO:11, SEQ ID NO:13, SEQ ID NO:15, and SEQ ID NO:17, but which differin codon sequence from any one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, orSEQ ID NO:17 due to the degeneracy of the genetic code, are also anaspect of this invention. The degeneracy of the genetic code, whichallows different nucleic acid sequences to code for the same protein orpeptide, is well known in the literature. See, e.g., U.S. Pat. No.4,757,006 to Toole et al. at Col. 2, Table 1.

Although nucleotide sequences which encode DsrA and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring DsrA under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding DsrA or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding DsrA and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

The invention also encompasses production of DNA sequences, or fragmentsthereof, which encode DsrA and its derivatives, entirely by syntheticchemistry. After production, the synthetic sequence may be inserted intoany of the many available expression vectors and cell systems usingreagents that are well known in the art. Moreover, synthetic chemistrymay be used to introduce mutations into a sequence encoding DsrA or anyfragment thereof.

Knowledge of the nucleotide sequence as disclosed herein in SEQ ID NO:1,SEQ ID NO:3. SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:15, and SEQ ID NO:17, can be used to generatehybridization probes which specifically bind to the DNA of the presentinvention or to mRNA to determine the presence of amplification oroverexpression of the proteins of the present invention.

The production of cloned genes, recombinant DNA, vectors, transformedhost cells, proteins and protein fragments by genetic engineering iswell known. See, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual (Cold Spring Harbor, N.Y. Cold Spring Harbor Laboratory (1989)),as well as U.S. Pat. No. 4,761,371 to Bell et al. at Col. 6 line 3 toCol. 9 line 65; U.S. Pat. No. 4,877,729 to Clark et al. at Col. 4 line38 to Col. 7 line 6; U.S. Pat. No. 4,912,038 to Schilling at Col. 3 line26 to Col. 14 line 12; and U.S. Pat. No. 4,879,224 to Wallner at Col. 6line 8 to Col. 8 line 59. (Applicant specifically intends that thedisclosure of all patent references cited herein be incorporated hereinin their entirety by reference).

Methods for DNA sequencing which are well known and generally availablein the art may be used to practice any of the embodiments of theinvention. The methods may employ such enzymes as the Klenow fragment ofDNA polymerase I, SEQUENASE® (US Biochemical Corp. Cleveland, Ohio), Taqpolymerase (Perkin Elmer), thermostable T7 polymerase (Amersham,Chicago, Ill.), or combinations of polymerases and proofreadingexonucleases such as those found in the ELONGASE Amplification Systemmarketed by Gibco/BRL (Gaithersburg, Md.). Preferably, the process isautomated with machines such as the Hamilton Micro Lab 2200 (Hamilton,Reno, Nev.), Peltier Thermal Cycler (PTC200; MJ Research, Watertown,Mass.) and the ABI Catalyst and 373 and 377 DNA Sequencers (PerkinElmer).

The nucleic acid sequences encoding DsrA may be extended utilizing apartial nucleotide sequence and employing various methods known in theart to detect upstream sequences such as promoters and regulatoryelements. For example, one method which may be employed.“restriction-site” PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar. G. PCR Method Applic.2,318–322 (1993)). In particular, genomic DNA is first amplified in thepresence of primer to a linker sequence and a primer specific to theknown region. The amplified sequences are then subjected to a secondround of PCR with the same linker primer and another specific primerinternal to the first one. Products of each round of PCR are transcribedwith an appropriate RNA polymerase and sequenced using reversetranscriptase.

A vector, as defined herein, is a replicable DNA construct. Vectors,such as plasmids, are used herein either to amplify DNA encoding theproteins of the present invention or to express the proteins of thepresent invention. An expression vector is a replicable DNA construct inwhich a DNA sequence encoding the proteins of the present invention isoperably linked to suitable control sequences capable of effecting theexpression of proteins of the present invention in a suitable host. Theneed for such control sequences will vary depending upon the hostselected and the transformation method chosen. Generally, controlsequences include a transcriptional promoter, an optional operatorsequence to control transcription, a sequence encoding suitable mRNAribosomal binding sites, and sequences which control the termination oftranscription and translation. Amplification vectors do not requireexpression control domains. All that is needed is the ability toreplicate in a host, usually conferred by an origin of replication, anda selection gene to facilitate recognition of transformants.

Vectors, as used herein, include plasmids, viruses (e.g., adenovirus,cytomegalovirus), phage, retroviruses and integratable DNA fragments(i.e., fragments integratable into the host genome by recombination).The vector replicates and functions independently of the host genome, ormay, in some instances, integrate into the genome itself. Expressionvectors preferably contain a promoter and RNA binding sites which areoperably linked to the gene to be expressed and are operable in the hostorganism.

DNA regions are operably linked or operably associated when they arefunctionally related to each other. For example, a promoter is operablylinked to a coding sequence if it controls the transcription of thesequence; a ribosome binding site is operably linked to a codingsequence if it is positioned so as to permit translation. Generally,operably linked means contiguous and, in the case of leader sequences,contiguous and in reading phase.

Transformed host cells are cells which have been transformed ortransfected with vectors containing DNA coding for proteins of thepresent invention need not express protein. Suitable host cells includeprokaryotes, yeast cells, or higher eukaryotic organism cells.Prokaryote host cells include gram negative or gram positive organisms,for example Escherichia coli (E. coli) or Bacilli. Higher eukaryoticcells include established cell lines of mammalian origin as describedbelow. Exemplary host cells are E. coli W3110) (ATCC 27.325), E. coli B,E. coli X1776 (ATCC 31,537), E. coli 294 (ATCC 31,446). A broad varietyof suitable prokaryotic and microbial vectors are available. E. coli istypically transformed using a derivative of the plasmid pBR322. SeeBolivar et al., Gene 2, 95 (1977). Promoters most commonly used inrecombinant microbial expression vectors include the beta-lactamase(penicillinase) and lactose promoter systems (Chang et al., Nature 275,615 (1978); and Goeddel et al., Nature 281, 544 (1979), a tryptophan(trp) promoter system (Goeddel et al., Nucleic Acids Res. 8, 4057 (1980)and EPO App. Publ. No. 36,776) and the tac promoter (H. De Boer et al.,Proc. Natl. Acad. Sci. USA 80, 21 (1983). The promoter andShine-Dalgarno sequence (for prokaryotic host expression) are operablylinked to the DNA of the present invention, i.e., they are positioned soas to promote transcription of the messenger RNA from the DNA.

Expression vectors should contain a promoter which is recognized by thehost organism. This generally means a promoter obtained from theintended host. While these are commonly used, other microbial promotersare suitable. Details concerning nucleotide sequences of many have beenpublished, enabling a skilled worker to operably ligate them to DNAencoding the protein in plasmid or viral vectors (Siebenlist et al.,Cell 20, 269 (1980). The promoter and Shine-Dalgarno sequence (forprokaryotic host expression) are operably linked to the DNA encoding thedesired protein, i.e., they are positioned so as to promotetranscription of the protein messenger RNA from the DNA.

Eukaryotic microbes such as yeast cultures may be transformed withsuitable protein-encoding vectors. See e.g., U.S. Pat. No. 4,745,057.Saccharomyces cerevisiae is the most commonly used among lowereukaryotic host microorganisms, although a number of other strains arecommonly available. Yeast vectors may contain an origin of replicationfrom the 2 micron yeast plasmid or an autonomously replicating sequence(ARS), a promoter, DNA encoding the desired protein, sequences forpolyadenylation and transcription termination, and a selection gene. Anexemplary plasmid is YRp7, (Stinchcomb et al., Nature 282, 39 (1979);Kingsman et al., Gene 7, 141 (1979); Tschemperet al., Gene 10,157(1980)). This plasmid contains the trp1 gene, which provides aselection marker for a mutant strain of yeast lacking the ability togrow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones,Genetics 85, 12 (1977)). The presence of the trp1 lesion in the yeasthost cell genome then provides an effective environment for detectingtransformation by growth in the absence of tryptophan. Suitablepromoting sequences in yeast vectors include the promoters formetallothionein, 3-phospho-glycerate kinase (Hitzeman et al., J. Biol.Chem. 255, 2073 (1980) or other glycolytic enzymes (Hess et al., J. Adv.Enzyme Reg. 7, 149 (1968); and Holland et al., Biochemistry 17, 4900(1978)), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase. Suitable vectors and promoters for use in yeast expressionare further described in R. Hitzeman et al., EPO Publn, No. 73,657.

Cultures of cells derived from multicellular organisms are a desirablehost for recombinant protein synthesis. In principal, any highereukaryotic cell culture is workable, whether from vertebrate orinvertebrate culture, including insect cells. Propagation of such cellsin cell culture has become a routine procedure. See Tissue Culture,Academic Press, Kruse and Patterson, editors (1973). Examples of usefulhost cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO)cell lines, and WI138, BHK, COS-7, CV, and MDCK cell lines. Expressionvectors for such cells ordinarily include (if necessary) an origin ofreplication, a promoter located upstream from the gene to be expressed,along with a ribosome binding site, RNA splice site (ifintron-containing genomic DNA is used), a polyadenylation site, and atranscriptional termination sequence.

The transcriptional and translational control sequences in expressionvectors to be used in transforming vertebrate cells are often providedby viral sources. For example, commonly used promoters are derived frompolyoma, Adenovirus 2, and Simian Virus 40 (SV40). See, e.g., U.S. Pat.No. 4,599.308. The early and late promoters are useful because both areobtained easily from the virus as a fragment which also contains theSV40 viral origin of replication. See Fiers et al., Nature 273, 113(1978). Further, the protein promoter, control and/or signal sequences,may also be used, provided such control sequences are compatible withthe host cell chosen.

An origin of replication may be provided either by construction of thevector to include an exogenous origin, such as may be derived from SV40or other viral source (e.g. Polyoma. Adenovirus, VSV, or BPV), or may beprovided by the host cell chromosomal replication mechanism. If thevector is integrated into the host cell chromosome, the latter may besufficient.

Host cells such as insect cells (e.g., cultured Spodoptera frugiperdacells) and expression vectors such as the baculovirus expression vector(e.g. vectors derived from Autographa californica MNPV, Trichoplusia niMNPV. Rachiplusia ou MNPV, or Galleria ou MNPV) may be employed to makeproteins useful in carrying out the present invention, as described inU.S. Pat. Nos. 4,745,051 and 4.879,236 to Smith et al. In general, abaculovirus expression vector comprises a baculovirus genome containingthe gene to be expressed inserted into the polyhedrin gene at a positionranging from the polyhedrin transcriptional start signal to the ATGstart site and under the transcriptional control of a baculoviruspolyhedrin promoter.

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, sequences encoding DsrA may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain a viable virus which iscapable of expressing DsrA in infected host cells (Logan, J. and Shenk,T. (1984) Proc. Natl. Acad. Sci. 81:3655–3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells. Rather thanusing vectors which contain viral origins of replication, one cantransform mammalian cells by the method of cotransformation with aselectable marker and the chimeric protein DNA. An example of a suitableselectable marker is dihydrofolate reductase (DHFR) or thymidine kinase.See U.S. Pat. No. 4,399,216. Such markers are proteins, generallyenzymes, that enable the identification of transformant cells. i.e.cells which are competent to take up exogenous DNA. Generally,identification is by survival or transformants in culture medium that istoxic, or from which the cells cannot obtain critical nutrition withouthaving taken up the marker protein.

In general, those skilled in the art will appreciate that minordeletions or substitutions may be made to the amino acid sequences ofpeptides of the present invention without unduly adversely affecting theactivity thereof. Thus, peptides containing such deletions orsubstitutions are a further aspect of the present invention. In peptidescontaining substitutions or replacements of amino acids, one or moreamino acids of a peptide sequence may be replaced by one or more otheramino acids wherein such replacement does not affect the function ofthat sequence. Such changes can be guided by known similarities betweenamino acids in physical features such as charge density,hydrophobicity/hydrophilicity, size and configuration, so that aminoacids are substituted with other amino acids having essentially the samefunctional properties. For example: Ala may be replaced with Val or Ser;Val may be replaced with Ala, Leu, Met, or lie, preferably Ala or Leu;Leu may be replaced with Ala, Val or lie, preferably Val or Ile; Gly maybe replaced with Pro or Cys, preferably Pro; Pro may be replaced withGly, Cys, Ser, or Met, preferably Gly. Cys, or Ser; Cys may be replacedwith Gly, Pro, Ser, or Met, preferably Pro or Met; Met may be replacedwith Pro or Cys, preferably Cys; His may be replaced with Phe or Gln,preferably Phe; Phe may be replaced with His, Tyr, or Trp, preferablyHis or Tyr; Tyr may be replaced with His, Phe or Trp, preferably Phe orTrp; Trp may be replaced with Phe or Tyr, preferably Tyr; Asn may bereplaced with Gln or Ser, preferably Gln; KGln may be replaced with His,Lys, Glu, Asn, or Ser, preferably Asn or Ser; Ser may be replaced withGln, Thr, Pro, Cys or Ala; Thr may be replaced with Gln or Ser,preferably Ser; Lys may be replaced with Gln or Arg; Arg may be replacedwith Lys, Asp or Glu, preferably Lys or Asp; Asp may be replaced withLys, Arg, or Glu, preferably Arg or Glu; and Glu may be replaced withArg or Asp, preferably Asp. Once made, changes can be routinely screenedto determine their effects on function with enzymes.

As noted above, the present invention provides isolated and purifiedDsrA proteins, such as mammalian (or more preferably human) DsrA. Suchproteins can be purified from host cells which express the same, inaccordance with known techniques, or even manufactured synthetically.

Nucleic acids of the present invention, constructs containing the sameand host cells that express the encoded proteins are useful for makingproteins of the present invention. Specific initiation signals may alsobe used to achieve more efficient translation of polynucleotidesequences encoding DsrA. Such signals include the ATG initiation codonand adjacent sequences. In cases where sequences encoding DsrA, itsinitiation codon, and upstream sequences are inserted into theappropriate expression vector, no additional transcriptional ortranslational control signals may be needed. However, in cases whereonly coding sequence, or a fragment thereof, is inserted, exogenoustranslational control signals including the ATG initiation codon shouldbe provided. Furthermore, the initiation codon should be in the correctreading frame to ensure translation of the entire insert. Exogenoustranslational elements and initiation codons may be of various origins,both natural and synthetic. The efficiency of expression may be enhancedby the inclusion of enhancers which are appropriate for the particularcell system which is used, such as those described in the literature(Scharf, D. et al. Results Probl. Cell Differ. 20,125–162(1994)). Inaddition, a host cell strain may be chosen for its ability to modulatethe expression of the inserted sequences or to process the expressedprotein in the desired fashion. Such modifications of the polypeptideinclude, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells which have specific cellular machineryand characteristic mechanisms for post-translational activities (e.g.,CHO, HeLa, MDCK, HEK293, and WI38), are available from the American TypeCulture Collection (ATCC; Manassas, Va.) and may be chosen to ensure thecorrect modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressDsrA may be transformed using expression vectors which may contain viralorigins of replication and/or endogenous expression elements and aselectable marker gene on the same or on a separate vector. Followingthe introduction of the vector, cells may be allowed to grow for 1–2days in an enriched media before they are switched to selective media.The purpose of the selectable marker is to confer resistance toselection, and its presence allows growth and recovery of cells whichsuccessfully express the introduced sequences. Resistant clones ofstably transformed cells may be proliferated using tissue culturetechniques appropriate to the cell type. Any number of selection systemsmay be used to recover transformed cell lines. These include, but arenot limited to, the herpes simplex virus thymidine kinase (Wigler, M. etal. Cell 11, 223–32 (1977)) and adenine phosphoribosyltransferase (Lowy,I. et al., Cell 22, 817–23 (1980)) genes which can be employed in tk- oraprt-cells, respectively. Also, antimetabolite or antibiotic resistancecan be used as the basis for selection; for example, dhfr which confersresistance to methotrexate (Wigler, M. et al., Proc. Natl. Acad. Sci.77, 3567–70 (1980)); npt, which confers resistance to theaminoglycosides neomycin and G-418 (Colbere-Garapin, F. et al., J. Mol.Biol. 150,1–14 (1981)) and als or pat, which confer resistance tochlorsulfuron and phosphinotricin acetyltransferase, respectively(Murry, supra). Additional selectable genes have been described, forexample, trpB, which allows cells to utilize indole in place oftryptophan, or hisD, which allows cells to utilize histinol in place ofhistidine (Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad.Sci. 85:8047–51). Recently, the use of visible markers has gainedpopularity with such markers as anthocyanins. β-glucuronidase and itssubstrate GUS, and luciferase and its substrate luciferin, being widelyused not only to identify transformants, but also to quantify the amountof transient or stable protein expression attributable to a specificvector system (Rhodes, C. A. et al. (1995) Methods Mol. Biol.55:121–131).

Although the presence/absence of marker gene expression suggests thatthe gene of interest (i.e., dsrA) is also present, its presence andexpression may need to be confirmed. For example, if the sequenceencoding DsrA is inserted within a marker gene sequence, transformedcells containing sequences encoding DsrA can be identified by theabsence of marker gene function. Alternatively, a marker gene can beplaced in tandem with a sequence encoding DsrA under the control of asingle promoter. Expression of the marker gene in response to inductionor selection usually indicates expression of the tandem gene as well.

Alternatively, host cells which contain the nucleic acid sequenceencoding DsrA and express DsrA may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA—DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein.

As explained further herein, proteins of the present invention areuseful as immunogens for making antibodies as described herein, andthese antibodies and proteins provide a “specific binding pair.” Suchspecific binding pairs are useful as components of a variety ofimmunoassays and purification techniques, as is known in the art. Theproteins of the present invention are of known amino acid sequence asdisclosed herein, and hence are useful as molecular weight markers indetermining the molecular weights of proteins of unknown structure.

The presence of polynucleotide sequences encoding DsrA can be detectedby DNA—DNA or DNA-RNA hybridization or amplification using probes orfragments or fragments of polynucleotides encoding DsrA. Nucleic acidamplification based assays involve the use of oligonucleotides oroligomers based on the sequences encoding DsrA to detect transformantscontaining DNA or RNA encoding DsrA.

A variety of protocols for detecting and measuring the expression ofDsrA, using either polyclonal or monoclonal antibodies specific for theprotein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson DsrA is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211–1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding DsrA includeoligolabeling, nick translation, end-labeling or PCR amplification usinga labeled nucleotide. Alternatively, the sequences encoding DsrA, or anyfragments thereof may be cloned into a vector for the production of anmRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3, or SP6 and labelednucleotides. These procedures may be conducted using a variety ofcommercially available kits (Pharmacia & Upjohn, (Kalamazoo, Mich.);Promega (Madison Wis.); and U.S. Biochemical Corp., Cleveland. Ohio)).Suitable reporter molecules or labels, which may be used for ease ofdetection, include radionuclides, enzymes, fluorescent,chemiluminescent, or chromogenic agents as well as substrates,cofactors, inhibitors, magnetic particles, and the like.

Host cells transformed with nucleotide sequences encoding DsrA may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a transformedcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeDsrA may be designed to contain signal sequences which direct secretionof DsrA through a prokaryotic or eukaryotic cell membrane. Otherconstructions may be used to join sequences encoding DsrA to nucleotidesequence encoding a polypeptide domain which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen, San Diego, Calif.) between the purificationdomain and DsrA may be used to facilitate purification. One suchexpression vector provides for expression of a fusion protein containingDsrA and a nucleic acid encoding 6 histidine residues preceding athioredoxin or an enterokinase cleavage site. The histidine residuesfacilitate purification on IMAC (immobilized metal ion affinitychromatography) as described in Porath, J. et al., Prot. Exp. Purif. 3,263–281 (1992)) while the enterokinase cleavage site provides a meansfor purifying DsrA from the fusion protein. A discussion of vectorswhich contain fusion proteins is provided in Kroll, D. J. et al., DNACell Biol. 12, 441–453 (1993)).

In addition to recombinant production, fragments of DsrA may be producedby direct peptide synthesis using solid-phase techniques (Merrifield J.J. Am. Chem. Soc. 85, 2149–2154 (1963)). Protein synthesis may beperformed using manual techniques or by automation. Automated synthesismay be achieved, for example, using Applied Biosystems 431A PeptideSynthesizer (Perkin Elmer). Various fragments of DsrA may be chemicallysynthesized separately and combined using chemical methods to producethe full length molecule.

Antibodies that specifically bind DsrA (i.e., antibodies which bind to asingle antigenic site or epitope on the proteins) are useful for avariety of diagnostic and therapeutic purposes. Antibodies to DsrA maybe generated using methods that are well known in the art. Suchantibodies may include, but are not limited to, polyclonal, monoclonal,chimeric, single chain, Fab fragments, and fragments produced by a Fabexpression library. Neutralizing antibodies, (i.e., those which inhibitdimer formation) are especially preferred for therapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith DsrA or any fragment or oligopeptide thereof which has immunogenicproperties. Depending on the host species, various adjuvants may be usedto increase immunological response. Such adjuvants include, but are notlimited to, Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

Monoclonal antibodies to DsrA may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique (Kohler, G. et al. (1975) Nature 256:495–497; Kozbor, D. etal. (1985) J. Immunol. Methods 81:31–42; Cote, R. J. et al. (1983) Proc.Natl. Acad. Sci. 80:2026–2030; Cole, S. P. et al. (1984) Mol. Cell Biol.62:109–120). Briefly, the procedure is as follows: an animal isimmunized with DsrA or immunogenic fragments or conjugates thereof. Forexample, haptenic oligopeptides of DsrA can be conjugated to a carrierprotein to be used as an immunogen. Lymphoid cells (e.g. spleniclymphocytes) are then obtained from the immunized animal and fused withimmortalizing cells (e.g. myeloma or heteromyeloma) to produce hybridcells. The hybrid cells are screened to identify those which produce thedesired antibody.

Human hybridomas which secrete human antibody can be produced by theKohler and Milstein technique. Although human antibodies are especiallypreferred for treatment of human, in general, the generation of stablehuman—human hybridomas for long-term production of human monoclonalantibody can be difficult. Hybridoma production in rodents, especiallymouse, is a very well established procedure and thus, stable murinehybridomas provide an unlimited source of antibody of selectcharacteristics. As an alternative to human antibodies, the mouseantibodies can be converted to chimeric murine/human antibodies bygenetic engineering techniques. See V. T. Oi et al., Bio Techniques4(4):214–221 (1986); L. K. Sun et al., Hybridoma 5 (1986).

The monoclonal antibodies specific for DsrA epitopes can be used toproduce anti-idiotypic (paratope-specific) antibodies. See e.g.,McNamara et al., Dec. 14, 1984, Science, page 1325; Kennedy, R. C. etal., (1986) Science 232:220. These antibodies resemble the DsrA epitopeand thus can be used as an antigen to stimulate an immune responseagainst H. ducreyi.

In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81, 6851–6855; Neuberger, M. S. et al. (1984) Nature312:604–608; Takeda, S. et al. (1985) Nature 314:452–454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceDsrA-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobin libraries (BurtonD. R. (1991) Proc. Natl. Acad. Sci. 88,11120–3).

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature(Orlandi, R. et al., Proc. Natl. Acad. Sci. 86, 3833–3837 (1989));Winter, G. et al., (1991) Nature 349, 293–299 (1991)).

Antibody fragments which contain specific binding sites for DsrA mayalso be generated. For example, such fragments include, but are notlimited to, the F(ab′)₂ fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse, W. D. et al. (1989) Science 254:1275–1281).

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between DsrA and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering DsrA epitopes is preferred, but a competitivebinding assay may also be employed (Maddox, supra).

Antibodies may be conjugated to a solid support suitable for adiagnostic assay (e.g., beads, plates, slides or wells formed frommaterials such as latex or polystyrene) in accordance with knowntechniques, such as precipitation. Antibodies may likewise be conjugatedto detectable groups such as radiolabels (e.g., ³⁵S, ¹²⁵I, ¹³¹I), enzymelabels (e.g., horseradish peroxidase, alkaline phosphatase), andfluorescent labels (e.g., fluorescein) in accordance with knowntechniques.

The proteins and peptides of this invention may be used as antigens inimmunoassays for the detection of H. ducreyi in various tissues and bodyfluids e.g., blood, spinal fluid, sputum, etc. A variety of immunoassaysystems may be used. These include: radio-immunoassays, ELISA assays,“sandwich” assays, precipitin reactions, gel diffusion precipitinreactions, immunodiffusion assays, agglutination assays, fluorescentimmunoassays, protein A immunoassays and immunoelectrophoresis assays.

In addition, nucleic acids having the nucleotide sequences of the geneencoding DsrA or any nucleotide sequences which hybridize therewith canbe used as probes in nucleic acid hybridization assays for the detectionof H. ducreyi in various tissues or body fluids of patients. The probesmay be used in any nucleic any type of hybridization assay including:Southern blots (Southern, 1975, J. Mol. Biol. 98:508); Northern blots(Thomas et al., 1980, Proc. Nat'l Acad. Sci. U.S.A. 77:5201–05); colonyblots (Grunstein et al., 1975. Proc. Nat'l Acad. Sci. U.S.A.72:3961–65), etc. Stringency of hybridization can be varied depending onthe requirements of the assay. Assays for detecting the polynucleotidesencoding DsrA in a cell, or the extent of amplification thereof,typically involve, first, contacting the cells or extracts of the cellscontaining nucleic acids therefrom with an oligonucleotide thatspecifically binds to DsrA polynucleotide as given herein (typicallyunder conditions that permit access of the oligonucleotide tointracellular material), and then detecting the presence or absence ofbinding of the oligonucleotide thereto. Again, any suitable assay formatmay be employed (see, e.g., U.S. Pat. No. 4,358,535 to Falkow et al.;U.S. Pat. No. 4,302,204 to Wahl et al.; U.S. Pat. No. 4,994,373 toStavrianopoulos et al; U.S. Pat. No. 4,486,539 to Ranki et al.; U.S.Pat. No. 4,563,419 to Ranki et al.; and U.S. Pat. No. 4,868,104 to Kurnet al.) (the disclosures of which applicant specifically intends beincorporated herein by reference).

Kits for determining if a sample contains proteins of the presentinvention will include at least one reagent specific for detecting thepresence or absence of the protein. Diagnostic kits for carrying outantibody assays may be produced in a number of ways. In one embodiment,the diagnostic kit comprises (a) an antibody which binds proteins of thepresent invention conjugated to a solid support and (b) a secondantibody which binds proteins of the present invention conjugated to adetectable group. The reagents may also include ancillary agents such asbuffering agents and protein stabilizing agents, e.g., polysaccharidesand the like. The diagnostic kit may further include, where necessary,other members of the signal-producing system of which system thedetectable group is a member (e.g., enzyme substrates), agents forreducing background interference in a test, control reagents, apparatusfor conducting a test, and the like. A second embodiment of a test kitcomprises (a) an antibody as above, and (b) a specific binding partnerfor the antibody conjugated to a detectable group. Ancillary agents asdescribed above may likewise be included. The test kit may be packagedin any suitable manner, typically with all elements in a singlecontainer along with a sheet of printed instructions for carrying outthe test.

Antisense oligonucleotides and nucleic acids that express the same maybe made in accordance with conventional techniques. See, e.g. U.S. Pat.No. 5,023,243 to Tullis; U.S. Pat. No. 5,149,797 to Pederson et al. Thelength of the antisense oligonucleotide (i.e., the number of nucleotidestherein) is not critical so long as it binds selectively to the intendedlocation, and can be determined in accordance with routine procedures.In general, the antisense oligonucleotide will be from 8, 10 or 12nucleotides in length up to 20, 30, or 50 nucleotides in length. Suchantisense oligonucleotides may be oligonucleotides wherein at least one,or all, or the internucleotide bridging phosphate residues are modifiedphosphates, such as methyl phosphonates, methyl phosphonothioates,phosphoromorpholidates, phosphoropiperazidates and phosphoramidates. Forexample, every other one of the internucleotide bridging phosphateresidues may be modified as described. In another non-limiting example,such antisense oligonucleotides are oligonucleotides wherein at leastone, or all, of the nucleotides contain a 2′ loweralkyl moiety (e.g.,C₁–C₄, linear or branched, saturated or unsaturated alkyl, such asmethyl, ethyl, ethenyl, propyl, 1-propenyl, 2-propenyl, and isopropyl).For example, every other one of the nucleotides may be modified asdescribed. See also P. Furdon et al., Nucleic Acids Res. 17, 9193–9204(1989); S. Agrawal et al., Proc. Natl. Acad. Sci. USA 87, 1401–1405(1990); C. Baker et al., Nucleic Acids Res. 18, 3537–3543 (1990); B.Sproat et al., Nucleic Acids Res. 17, 3373–3386 (1989); R. Walder and J.Walder, Proc. Natl. Acad. Sci. USA 85, 5011–5015 (1988).

In another embodiment of the invention, DsrA, its catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, betweenDsrA and the agent being tested, may be measured.

Another technique for drug screening which may be used provides for highthroughput screening of compounds having suitable binding affinity tothe protein of interest as described in published PCT applicationWO84/03564. In this method, as applied to DsrA, large numbers ofdifferent small test compounds are synthesized on a solid substrate,such as plastic pins or some other surface. The test compounds arereacted with DsrA, or fragments thereof, and washed. Bound DsrA is thendetected by methods well known in the art. Purified DsrA can also becoated directly onto plates for use in the aforementioned drug screeningtechniques. Alternatively, non-neutralizing antibodies can be used tocapture the peptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding DsrA specificallycompete with a test compound for binding (DsrA. In this manner, theantibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with DsrA.

The proteins, peptides, polynucleotides and vectors comprising thepolynucleotides of the present invention may be used as immunogens invaccines against H. ducreyi, which vaccines are an aspect of the presentinvention. When used as an immunogen, it is not necessary to use theentire DsrA protein, although the entire DsrA protein may be used.Polypeptides, fragments, and/or antigenic determinants of DsrA may alsobe used as immunogens in the practice of the invention. The vaccines areused to prevent or reduce susceptibility to H. ducreyi infection.

The vaccines comprise an immunologically effective amount of theimmunogen in a pharmaceutically acceptable carrier. The combinedimmunogen and carrier may be an aqueous solution, emulsion, orsuspension. An immunologically effective amount is determinable by meansknown in the art without undue experimentation, given the teachingscontained herein. Pharmaceutically acceptable carriers are known tothose skilled in the art and include stabilizers, diluents, and buffers.Suitable stabilizers include carbohydrates, such as sorbitol, lactose,mannitol, starch, sucrose, dextran, and glucose and proteins, such asalbumin or casein. Suitable diluents include saline, Hanks BalancedSalts, and Ringers solution. Suitable buffers include an alkali metalphosphate, an alkali metal carbonate, or an alkaline earth metalcarbonate.

The immunogens of the invention are immunogenic without adjuvant,however adjuvants may increase immunoprotective antibody titers or cellmediated immunity response. Such adjuvants could include, but are notlimited to, Freund's complete adjuvant, Freund's incomplete adjuvant,aluminum hydroxide, aluminum phosphate, aluminum oxide or a compositionthat consists of a mineral oil, such as Marcol 52, or a vegetable oiland one or more emulsifying agents, dimethyldioctadecyl-ammoniumbromide, ADJUVAX (Alpha-Beta Technology), Inject Alum (Pierce),Monophosphoryl Lipid A (Ribi Immunochem Research), MPL+TDM (RibiImmunochem Research), TITERMAX (CytRx), toxins, toxoids, glycoproteins,lipids, glycolipids, bacterial cell walls, subunits (bacterial orviral), carbohydrate moieties (mono-, di-, tri- tetra-, oligo- andpolysaccharide) various liposome formulations or saponins. Otheradjuvants that may be included in vaccine compositions of the presentinvention include, but are not limited to: surface active substances(e.g., hexadecylamine, octadecylamine, octadecyl amino acid esters,lysolecithin, dimethyl-dioctadecylammonium bromide),methoxyhexadecylgylcerol, pluronic polyols; polyamines (e.g., pyran,dextransulfate, poly IC, CARBOPOL); and peptides (e.g., muramyldipeptide, dimethylglycine, tuftsin). The immunogen may also beincorporated into liposomes, or conjugated to polysaccharides and/orother polymers for use in a vaccine formulation. Combinations of variousadjuvants may be used with the conjugate to prepare the immunogenformulation. Exact formulation of the vaccine compositions will dependon the particular conjugate, the species to be immunized and the routeof administration.

The vaccines of the invention are prepared by techniques known to thoseskilled in the art, given the teachings contained herein. Generally, theimmunogens are mixed with the carrier to form a solution, suspension, oremulsion. One or more of the additives discussed above may be in thecarrier or may be added subsequently. The vaccine preparations may bedessicated, for example, by freeze drying for storage purposes. If so,they may be subsequently reconstituted into liquid vaccines by theaddition of an appropriate liquid carrier.

Any suitable vaccine and method of vaccination (i.e. immunization) knownin the art may be employed in carrying out the present invention, aslong as an active immune response against the antigen is elicited. Whenadministered according to the present invention, the vaccine induces anactive and protective immune response against unmodified cancer cells.Exemplary vaccination methods include, but are not limited to, “nakedDNA” vaccines, viral and bacterial vector vaccines, liposome associatedantigen vaccines, and peptide vaccines. Vaccines may be live vaccines,attenuated vaccines, killed vaccines, or subunit vaccines. Methods ofvaccinating animals and humans against immunogens are well-known in theart. See, e.g., S. Crowe et al. Infections of the Immune System, inBasic and Clinical Immunology, 697–715 (D. P. Stites & A. I. Terr. eds.,7th ed. 1991).

The vaccines of the present invention are administered to humans orother mammals, including bovine, ovine, caprine, equine, leporine,porcine, canine, feline and avian species, with humans beingparticularly preferred. The vaccines may administered to human children,including children younger than 18 months of age. Preferably, thevaccines of the present invention are administered to those subjectsthat are at particular risk of developing H. ducreyi infection (i.e.,subjects living in geographic locations where H. ducreyi is common).

The vaccines may be administered in one or more doses. The vaccines maybe administered by known routes of administration for this type ofvaccine, including parenteral administration, such as subcutaneous,intramuscular, or intravenous administration. Oral administration mayalso be used, including oral dosage forms which are enteric coated.

The schedule of administration of the vaccine may vary depending on thestrain of H. ducreyi being used, the age and/or condition of the subjectto be immunized, the particular formulation of the vaccine, and otherfactors known to those in the art. Subjects may receive a single dose,or may receive a booster dose or doses. Annual boosters may be used forcontinued protection.

The immunogens of this invention can be formulated as univalent andmultivalent vaccines. The immunogens (i.e., the protein DsrA) can bemixed, conjugated or fused with other antigens, including B or T cellepitopes of other antigens. In addition to its utility as a primaryimmunogen. DsrA can be used as a carrier protein to confer or enhanceimmunogenicity of other antigens.

When a haptenic peptide of DsrA is used, (i.e., a peptide which reactswith cognate antibodies, but cannot itself elicit an immune response),it can be conjugated to an immunogenic carrier molecule. For example, anoligopeptide containing one or more epitopes of DsrA may be haptenic.Conjugation to an immunogenic carrier can render the oligopeptideimmunogenic. Preferred carrier proteins for the haptenic peptides ofDsrA are tetanus toxin or toxoid, diphtheria toxin or toxoid and anymutant forms of these proteins such as CRM 197. Others include exotoxinA of Pseudomonas, heat labile toxin of E. coli and rotaviral particles(including rotavirus and VP6 particles). Alternatively, a fragment orepitope of the carrier protein or other immunogenic protein can be used,or example, the hapten can be coupled to a T cell epitope of a bacterialtoxin.

The peptides or proteins of this invention can be administered asmultivalent subunit vaccines in combination with other antigens of H.ducreyi. For example, they may be administered in conjunction witholigo- or polysaccharide capsular components of H. ducreyi such aspolyribosylribitolphosphate (PRP).

Peptides and proteins having epitopes of DsrA evoke bactericidalantibodies which may act synergistically in killing H. ducreyi withantibodies against other outer membrane proteins of H. ducreyi. Thus, inan embodiment of the invention. DsrA (or a peptide or protein having acommon epitope) is administered in conjunction with other outer membraneproteins of H. ducreyi (or peptides or proteins having epitopes thereof)to achieve a synergistic bactericidal activity. For combinedadministration with epitopes of other outer membrane proteins, the DsrApeptide can be administered separately, as a mixture or as a conjugateor genetic fusion peptide or protein. The conjugates can be formed bystandard techniques for coupling proteinaceous materials. Fusions can beexpressed from fused gene constructs prepared by recombinant DNAtechniques as described.

The immunogens of this invention can be administered as live vaccines.To this end, recombinant microorganisms are prepared that express thepeptides or proteins. The vaccine recipient is inoculated with therecombinant microorganism which multiplies in the recipient, expressesthe DsrA peptide or protein and evokes a immune response to H. ducreyi.Preferred live vaccine vectors are pox viruses such as vaccinia(Paoletti and Panicali. U.S. Pat. No. 4,603,112) and attenuatedSalmonella strains (Stocker, U.S. Pat. No. 4,550,081).

Live vaccines are particularly advantageous because they lead to aprolonged stimulus which can confer substantially long-lasting immunity.When the immune response is protective against subsequent H. ducreyiinfection, the live vaccine itself may be used in a preventative vaccineagainst H. ducreyi.

Multivalent live vaccines can be prepared from a single or a fewrecombinant microorganisms that express different epitopes of H.ducreyi. In addition, epitopes of other pathogenic microorganisms can beincorporated into the vaccine. For example, a vaccinia virus can beengineered to contain coding sequences for other epitopes in addition tothose of H. ducreyi. Such a recombinant virus itself can be used as theimmunogen in a multivalent vaccine. Alternatively, a mixture of vacciniaor other viruses, each expressing a different gene encoding fordifferent epitopes of outer membrane proteins of H. influenzae and/orepitopes of other disease causing organisms can be formulated as amultivalent vaccine.

An inactivated virus or bacterial vaccine may be prepared. Inactivatedvaccines are “dead” in the sense that their infectivity has beendestroyed, usually by chemical treatment (e.g., formaldehyde treatment).Ideally, the infectivity of the virus or bacteria is destroyed withoutaffecting the proteins which carry the immunogenicity of the vector. Inorder to prepare inactivated vaccines, large quantities of therecombinant vector expressing the desired epitopes are grown in cultureto provide the necessary quantity of relevant antigens. A mixture ofinactivated viruses or bacteria expressing different epitopes may beused for the formulation of “multivalent” vaccines. In certaininstances, these “multivalent” inactivated vaccines may be preferable tolive vaccine formulation because of potential difficulties arising frommutual interference of live viruses administered together. In eithercase, the inactivated virus or mixture of viruses should be formulatedin a suitable adjuvant in order to enhance the immunological response tothe antigens. Suitable adjuvants include: surface active substances,e.g., hexadecylamine, octadecyl amino acid esters, octadecylamine,lysolecithin, dimethyl-dioctadecylammonium bromide, N,N-dicoctadecyl-N′-N′bis (2-hydroxyethyl-propane diamine),methoxyhexadecylglycerol, and pluronic polyols; polyamines, e.g., pyran,dextransulfate, poly IC, CARBOPOL; peptides, e.g., muramyl dipeptide,dimethylglycine, tuftsin; oil emulsions; and mineral gels, e.g.,aluminum hydroxide, aluminum phosphate, etc.

One particularly preferred embodiment of the invention is an attenuatedvaccine comprising an H. ducreyi strain that does not express DsrA. TheH. ducreyi strains that do not express DsrA used in these vaccines maybe naturally occurring strains, or may be recombinant and/or isogenicmutants of H. ducreyi strains that do express the protein. Of theseattenuated vaccines, a vaccine comprising the H. ducreyi mutant strainFX517 described herein is most preferred.

The bactericidal antibodies induced by DsrA epitopes can be used topassively immunize an individual against H. ducreyi. Passiveimmunization confers short-term protection for a recipient by theadministration of the pre-formed antibody. Passive immunization can beused on an emergency basis for special risks, e.g. young childrenexposed to contact with subjects already afflicted with H. ducreyiinfection (chancroid).

In view of the foregoing description, the invention also comprises amethod for inducing an immune response to H. ducreyi in a mammal inorder to protect the mammal against infection by invasive ornon-invasive H. ducreyi. The method comprises administering animmunologically effective amount of the immunogens of the invention tothe host and, preferably, administering the vaccines of the invention tothe host.

The following Examples are provided to illustrate the present invention,and should not be construed as limiting thereof. Unless otherwise noted,all chemicals and reagents were from Sigma Chemicals (St. Louis. MO).Standard recombinant DNA methods were used as described in Sambrook etal. (supra) or following manufacturers instructions.

EXAMPLE 1 Materials and Methods: Bacterial Strains and Media

Bacterial strains used in the experiments described herein are shownbelow in Table 1. For routine growth. H. ducreyi was maintained onchocolate agar plates obtained from UNC Hospital Clinical MicrobiologyLab. This medium was prepared using Mueller Hinton base and contained nofetal calf serum. When 5% fetal calf serum was required for optimalgrowth (H. ducreyi strains CHIA and 1157), Gonococcal medium base (GCB)was used for preparation and instructions were followed (Difco).Antibiotics were used at the following concentrations for E. coli:ampicillin, 100 μg/ml; chloramphenicol, 30 μg/ml; kanamycin, 30 μg/ml;and streptomycin, 100 μg/ml. For H. ducreyi, antibiotics werechloramphenicol, 1 μg/ml or streptomycin, 100 μg/ml.

TABLE I Bacterial strains and plasmids Source/Reference/ Strain/PlasmidRelevant Genotype/Phenotype Isolated E. coli K-12 DH5αLMCR recA, gyrBBethesda Research Labs H. ducrevi 35000 wild type Stanley SpinolaIndiana Univ. FX516 35000 Co-integrate This work beta galactosidasepositive intermediate in FX517 construction, Cm^(r) FX517 35000 dsrA,Cm^(r) This work CIP542 (Canada) William Albritton CIP A77 Robert MunsonCIP 542 (CDC) Stephen Morse Centers for Disease Control H. ducrevi (10)obtained from Pat Totten CIP A75 Pasteur Institute CHIA VDRL HD167 VDRLV-1157 Seattle V-1168 Seattle M90-02 Bahamas 406 Mississippi 425Mississippi 54 Mississippi 010-2 Dominican Republic HD301 Thailand HD350Kenya Plasmids pCRII PCR cloning vector Invitrogen Kan^(r), Amp^(r)pUNCH 1248 dsrA PCR clone using This work primers 14 and 16 in pCRIIvector pLS88 Shuttle plasmid (9) Kan^(r), Str^(r), Sul^(r) pUNCH 1254dsrA subclone. ECoR1 This work fragment of pUNCH 1248 in EcoR1 of pLS88pUNCH 1255 mutagenized dsrA; This work pUNCH 1254 mutagenized with CATcassette from pNC40 Kan^(r), Cm^(r) This work pRSM1791 Mutagenesisplasmid (6) Beta gal^(r), Amp^(r) pUNCH 1256 pUNCH 1255 This work(Smal/HinClI/Klenow) into the NotI (Klenow) of pRSM1791 pUNCH 1260 dsrAPCR clone using This work primers 14 and 16 in pLSKS pNC40 source of CATcassette, (37) Amp^(r), Cm^(r)

EXAMPLE 2 Outer Membrane Isolation, Analysis, SDS-PAGE andImmunoblotting

Large scale cultures of H. ducreyi were performed in Fernbach flaskswith 1 liter of GCB-1 broth containing 5% fetal calf serum and 50 μg/mlheme (Elkins, C. Identification and purification of a conservedheme-regulated hemoglobin-binding outer membrane protein fromHaemophilus ducreyi. Infec Immun. 63, 1241–1245 (1995)). Cultures of E.coli were performed in LB broth or LB agar plates containing appropriateantibiotics. Outer membranes were harvested as previously described Id.Protein concentrations were determined using the BCA kit from Pierce(Rockford, Ill.). SDS-PAGE, and Western blotting were performed aspreviously described (11). The lipooligosaccharide (LOS) of H. ducreyiwas prepared using the method of Hitchcock and Brown (Hitchcock, P. G.,and Brown, T. M. Morphological heterogeneity among Salmonella LPSchemotypes, in silver-stained polyacrylamide gels. J. Bacteriol. 154,269–277 (1983). LOS was analyzed by SDS-PAGE and silver staining (Tsai,C. M. and Frasch, C. E., A sensitive silver stain for detectinglipopolysaccharides in polyacrylamide gels. Anal. Biochem. 155, 115–119(1982)) or Western blotting with Mab 3F11 (Apicella, M. A. et al.,Phenotypic variation in epitope expression of the Neisseria gonorrhoeaelipooligosaccharide. Infect Immun. 55:1755–1761 (1987).

EXAMPLE 3 N-Terminal Sequence Amino Acid (AA) Determination

The N-terminal aa sequence of DsrA was determined from strain 35000.Outer membranes were subjected to preparative SDS-PAGE and Westerntransfer to PVDF. The blot was stained temporarily with Ponceau Sprotein stain to locate the DsrA protein, which in strain 35000 migratesjust below the 30 kDa standard protein. Strips of the blot were probedwith anti-OpaF (generously provided by Janice Babcock and Richard Restof Hahnemann Medical College) of gonococcal strain FA1090 and Mab 5C9.Anti-OpaF, for unknown reasons, cross-reacts with DsrA and Mab 5C9reacts with a previously described H. ducreyi lipoprotein (termed Hlp)of similar molecular weight (18). These antibodies were used in order tounequivocally identify the proper band to sequence. The corresponding 30kDa-OpaF reactive band from the remainder of the Ponceau S stained blotwas sequenced. The sequence obtained from the 30 kDa band was QQPPKFAGVSSLYSYEYDYG KGKKTKSNEG (amino acid residues 22–51, SEQ ID NO:2). Thissequence did not match the processed mature, N-terminal sequence of Opaor Hlp 28 kDa (Hlp would be expected not to sequence, since it is alipoprotein). We concluded that these three proteins were distinct.

The antiserum to DsrA was produced as follows. Outer membranes from H.ducreyi strain 35000 were electophoresed on large preparative well 12%SDS-PAGE gels. The gel was briefly stained and the corresponding 30 kDaband excised and electroeluted using a CENTRILUTOR (Amicon) followingthe manufacturer's instructions. Mice were immunized a total of 3 timeswith 25 μg of gel purified protein per immunization. Freund's completeadjuvant was used for the first immunization and incomplete for theremainder.

EXAMPLE 4 Vector-Anchored PCR

Two degenerate oligonucleotides deduced from the N-terminal amino acidsequence (#6 and #7, FIG. 2) specifically hybridized to a 1.1 kb EcoR1genomic fragment (data not shown). Attempts to clone this fragment usingsize selected DNA using several plasmid vectors were unsuccessful.Therefore a series of three separate vector-anchored PCR strategies wereutilized to clone the dsrA structural gene, upstream flanking DNA anddownstream flanking DNA, respectively. The first vector-anchored PCR(FIG. 2, V-A PCR 1) used the ligation between the 1.1 kb EcoR1size-selected DNA and vector pBluescript as template and used 5′ primer#6 and vector primer KS as amplimers. An approximate 700 bp fragment wasamplified and preliminary sequence obtained. The N-terminal sequenceoriginally obtained from Edman degradation matched the deduced aminoacid sequence of the PCR product, but was not homologous to knownsequences in the data bases. In contrast, the C-terminus of the gene washomologous to UspA2 and YadA (see results below), this suggested thepossibility of PCR generated artifact(s). To rule out PCR artifactadditional PCR was performed. The primers used included 5′ primers #6, 8and 9 and 3′ primers 11 and 12. The latter 4 primers were derived fromthe DNA sequence obtained from the original anchored PCR product above(FIG. 2 and data not shown). Identically sized products from total H.ducreyi chromosomal DNA template (and the original anchored PCR product,the +control template were amplified) using 3′ primers from the regionwith homology to C-terminal YadA (primers #11 and #12) (data not shown).Furthermore, Southern hybridization of H. ducreyi chromosomal DNA probedwith oligos #6, #7, #8, #9, #11, #12 and the PCR product generated from#8 and #12 all specifically recognized the 1.1 kb band ECoR1 band (FIG.1 and data not shown). It was concluded that the N-terminal aa sequenceobtained from the 30 kDa protein is found on the same ORF that hasC-terminal homology to UspA2/YadA. These data established that the openreading frame (ORF) data were correct.

To obtain sequence upstream of the structural gene for dsrA, a secondvector-anchored PCR was used (FIG. 2, V-A PCR 2). Again, the templatewas the ligation between the 1.1 kb EcoR1 size-selected DNA and vectorpBluescript but the primers used were #12 and vector primer KS. A (1069)bp fragment which included the upstream EcoR1 site (FIG. 2.) wasamplified.

To obtain sequence downstream of the dsrA gene, a third vector-anchoredPCR was used (FIG. 2, V-A PCR 3). Southern hybridization identified anapproximately 4 kb Bgl II fragment which hybridized with dsrA probes andthere are no Bgl II sites in the 1.1 kb EcoR1 fragment. Fragments of 3–5kb Bgl II restricted chromosomal DNA were isolated and ligated to BamH1,shrimp alkaline phosphatase treated pMCL210 vector. The ligationreaction was ethanol precipitated and amplified using primers 10 andvector primer T7 (promoter), yielding an approximately 2.5 kb PCRproduct. The products of all three vector-anchored PCR reactions weresequenced with appropriate primers to obtain preliminary sequence andthese sequences confirmed one another (data not shown).

Commercially available PCR tubes (Ready to Go, Pharmacia) were utilizedfor PCR. Analytical PCR (25 ul final volume) utilized single tubeswhereas preparative PCR combined the “beads” from 4 tubes into singletube (100 ul final volume). The MgCl₂ concentration in all PCR reactionswas 4 mM. The first two vector anchored PCRs used 5 ul of ligation and25 pm of each primer. The conditions for PCR for first two vectoranchored PCRs were: hot start 5 min 94C, denature 94C; 1 minuteannealing, 50C, 1 minute; extension 72, 1 minute. The conditions for thethird PCR were identical except that the extension time was 3 min.

EXAMPLE 5 DNA Sequencing and Analysis

DNA sequence analysis was performed at the University of North Carolinaat Chapel Hill Automated Sequencing Facility utilizing Taq terminatorchemistry. The final sequences presented for strain 35000 in FIG. 2 andfor the other H. ducreyi strains in FIG. 9 was obtained from PCRproducts using primers #14 and 24 which flank the dsrA gene (FIG. 1).Both strands of the were completely sequenced. The sequence data wereassembled using the program AssemblyLIGN (IBI). The preliminary sequencefor the dsrA structural gene from 35000 obtained by vector-anchored PCRwas in complete agreement with the final sequence presented (FIG. 3).Amino acid alignments were done by Clustal in the program GeneJockeyII(Cambridge, UK) and PAM 250 setting. Bestfit (GCC Computer Group,Wisconsin) was used to generate similarity and identity scores using agap weight of 8.

EXAMPLE 6 Plasmid Constructions

Plasmid pUNCH 1248 was constructed by PCR. A 900 bp fragment wasamplified from H. ducreyi strain 35000 using primers 14 and 16 (FIG. 2),using the conditions described above for the first two vector anchoredPCRs. The product was ligated to pCRII following the manufacturer'sdirections, transformed into E. coli DH5a and recombinants identified byrestriction analysis. E. coli harboring pUNCH 1248 grew poorly, waspropagated only on agar plates to reduce the possibility ofmutation/deletion, and gave rise to an occasional larger colony.Subclone 1254 was constructed by isolating the EcoR1 fragment of pUNCH1248 and ligation into EcoR1 restricted pLS88. dsrA of pUNCH 1254 wasmutagenized by insertion of a CAT (Chloramphenicol Acetyl Transferase)into the open reading frame to construct pUNCH 1255. To perform this, aCAT cassette (a BglII fragment from pNC40 was treated with Klenow tofill-in the ends) was ligated into the NdeI site of pUNCH 1254(previously treated with Klenow to produce blunt ends), pUNCH 1256 wasconstructed by moving the insert from pUNCH 1255 (containing mutagenizeddsrA) into plasmid pRSM1791 for subsequent mutagenesis. This was done byisolation of a SmaI to HinCII fragment of pUNCH 1255, Klenow treatmentand ligation into the NotI site of pRSM1791 previously treated withKlenow. Transformation of an E. coli host was performed and selectionusing Amp and Cm yielded pUNCH 1256.

EXAMPLE 7 Construction and Characterization of an H. ducreyi DsrA Mutant

An isogenic mutant (FX517, Table 1) was constructed by allelicreplacement of the wild-type locus of strain 35000 with the mutation inpUNCH 1256 using a previous system of mutagenesis described by Bozue etal (Bozue, J. A. et al.; Facile construction of mutations in Haemophilusducreyi using lacZ as a counter-selectable marker. FEMS MicrobiologyLetters. 164, 269–73 (1998)). In this procedure, a mutagenized copy ofthe locus (containing a chloramphenicol (Cm or CAT) cassette) wassubcloned into a plasmid able to express lacZ (pUNCH 1256). H. ducreyiwere electroporated and Cm^(r) transformants selected (Elkins et al.,Characterization of the hgbA locus of Haemophilus ducreyi Infect Immun.63, 2194–2200 (1995); Hansen, E. J. et al., Use of electroporation toconstruct isogenic mutants of Haemophilus ducreyi. J. Bacteriol. 174,5442–9 (199)). These transformants putatively contained the entireplasmid integrated due to a single crossover event (as exemplified byFX516, Table 1). Individual transformants were streaked onto Cm mediumcontaining X-gal. Since the product of X-gal is highly toxic to H.ducreyi the co-integrates grow as tiny blue colonies. The loss of theX-gal sequences and neighboring wild type allele via a resolution of theco-integrate results in only the mutant allele being retained(exemplified by FX 517, Table 1). These H. ducreyi mutants grew asnormal-sized white colonies on the medium containing Cm and X-galsimilar to other H. ducreyi mutants containing CAT cassettes (Elkins, C.et al., Characterization of the hgba locus of Haemophilus ducreyi.Infect Immun. 63, 2194–2200 (1995) Elkins, C. et al., Role of theHaemophilus ducreyi Ton system in internalization of heme fromhemoglobin. Infection & Immunity 66,151–60 (1998); Thomas, C. et al.,Cloning and characterization of tdhA, a locus encoding a TonB-dependentheme receptor from Haemophilus ducreyi. Infect Immun. 66, 1–9 (1998))and data not shown.

Southern blot and PCR analysis was used to confirm that an allelicreplacement occurred in the generation of H. ducreyi mutant FX517.Chromosomal DNA was isolated from strains 35000. FX516, and FX517,digested with HinCII and subjected to electrophoresis and bidirectionaltransfer. The two blots were probed with either the PCR product ofoligos 14 and 16 or the Bgl II CAT fragment from pUNCH 40.Digoxigenin-labeled, bound probe was detected with alkaline phosphataselabeled anti-digoxigenin antibody (Boehringer Mannheim) followed bydetection with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolylphosphate (NBT/BCIP). PCR confirmation of the mutant utilized primers 14and 16 which flank the NdeI site (CAT cassette) used for genedisruption.

EXAMPLE 8 Complementation of FX517 and Other DsrA Mutants in Trans

To rule out that the serum susceptibility of dsrA mutant FX517 was dueto a mutation elsewhere on the chromosome or polar downstream effects,complementation in trans was performed. Briefly, we PCR amplified thedsrA and surrounding locus, using primers 14 and 24 (FIG. 2), Klenowtreated the PCR product, and restricted the PCR product with HinDIII(which restricts just downstream of dsrA, FIG. 2). After gelpurification, the PCR product was ligated into SmaI/HinDIII restrictedpLSKS (Wood, G. E. et al., Target and cell range of the Haemophilusducreyi hemolysin and its involvement in invasion of human epithelialcells. Infect and Immun. In Press.) The ligation was ethanolprecipitated and H. ducreyi strain FX517 electroporated. Streptomycinresistant colonies were screened for production of DsrA by Westernblotting and confirmed by restriction analysis. One experimentaltransformant, pUNCH 1260dsrA, and one vector transformant were selectedfor further study, pUNCH 1260 and the vector pLSKS (negative control)were then electroporated into the three naturally occurring dsrA mutants(CIP A75, CIP A77, CIP 542 (Can), Table 1).

EXAMPLE 9 Serum Susceptibility

The resistance of H. ducreyi to normal human serum was performed aspreviously described (Odumeru; Carbonetti) with the followingmodifications: An 18–24 hour culture of H. ducreyi from chocolate agarplates was scraped into GCB broth to an OD600 of 0.2. A 10⁻⁴ to 10⁻⁵dilution was made (approximately 1000 CFU/ml, depending on the strain)and mixed with pooled fresh normal human serum (NHS) or heat inactivatedNHS (56° C., 30 min) to a final concentration of 25 or 50% NHS. Afterincubation for 45 minutes at 35° C. in 5% CO₂, 100 μl aliquots wereplated onto chocolate agar plates and viable counts performed after 48hours. Data are expressed as percent survival in the fresh NHS ascompared to survival in heat-inactivated NHS (number of CFU survivors infHNS/number of survivors in heated NHS X 100). Strains containing pUNCH1260 or PLSKS were propagated and plated on chocolate agar containingstreptomycin at 100 μg/ml.

EXAMPLE 10 Identification of a 30 kDa Protein Involved in SerumResistance

During the course of studies characterizing the H. ducreyi interactionwith PMNs, a series of Western blots was performed using variousantibodies to the Opa proteins from gonococci. It was found that apolyclonal antiserum to OpaF of gonococcal strain FA1090 reacted at adilution of 1:5000 with a protein (DsrA) that varied between 28 and 35kDa in a panel of strains (data not shown). One strain, CIPA75, did notreact. CIPA75 was of interest because it had previously been shown to beavirulent in the chilled rabbit model of infection, to be serumsusceptible, to exhibit reduced adherence to HEp-2 cells and to have atruncated LOS (Odumeru, J. A. et al, Role of lipopolysaccharide andcomplement in susceptibility of Haemophilus ducreyi to human serum.Infect Immun. 50,495–9 (1985); Rice, P. A., Molecular basis for serumresistance in Neisseria gonorrhoeae. Clinical Microbiology Reviews. 2,S112–7 (1989). Specific antisera to DsrA were generated using DsrApurified by preparative SDS-PAGE and electroelution of outer membranesfrom H. ducreyi strain 35000. Western blots of several geographicallydiverse lab and clinical isolates were probed with anti-DsrA (FIG. 1).This was done to confirm that the previous cross-reactivity seen withthe anti-OpaF serum was due to the presence of DsrA and to ascertainwhat percentage of strains expressed dsrA. The proteins recognized inthe DsrA Western blot (FIG. 1) and the OpaF Western blot (data notshown) appeared to be identical. Most strains in FIG. 1 expressed animmunoreactive protein, except for the previously reported avirulentstrains CIP A75, CIP A77 (25–27) and CIP542 (Can., obtained from Canada)(Alfa, M. J. et al., Use of tissue culture and animal models to identifyvirulence-associated traits of Haemophilus ducreyi. Infection & Immunity63:1754–61 (1995)). In contrast virulent CIP 542 (CDC), obtained fromthe CDC and previously shown to cause a laboratory acquired infection(Trees, D. L. et al., Laboratory-acquired infection with Haemophilusducreyi type strain CIP 542. Med Microbiol 330–337 (1992)), expresseddsrA. Previous studies documented that virulent H. ducreyi strains areserum resistant. We performed serum susceptibility studies of selectedH. ducreyi strains which did and did not express dsrA and these resultsare summarized at the bottom of FIG. 1. For the purposes of this study,we arbitrarily termed a strain serum resistant if there were more than10% survivors when exposed to 50% fNHS serum as compared to NHS. Thespecific percent survivors (+/−sd) for each of the strains tested inFIG. 1 are: 35000, 79%; CIP A75; CIP A77; CIP 542 (Can); CIP 542 (CDC);CHIA; V-1157; M90-02; and 406. Thus, in these initial studies there wasa correlation between strains tested which expressed detectable dsrA andserum resistance. This correlation between the lack of expression ofdsrA and serum susceptibility in the dsrA mutant strains, some of whichalso had LOS alterations, could merely be coincidental. Thereforeadditional molecular studies were performed, culminating in thegeneration of an isogenic dsrA mutant for biological studies.

EXAMPLE 11 Molecular Studies

Through a series of experiments involving Western blotting,immunoprecipitation and finally N-terminal amino acid sequencing, it wasdetermined that the DsrA protein was not the same as the previouslydescribed 28 kDa lipoprotein termed Hlp (17) (data not shown). TheN-terminal amino acid sequence of the immunoreactive DsrA 30 kDa proteinof strain 35000 was found to be: QQPPKFAGVS SLYSYEYDYG KGKKTKSNEG (aminoacid residues 22–51, SEQ ID NO:2). No known homologies were initiallydetected when this peptide sequence was searched against GENBANK,including gonococcal Opa proteins.

Two degenerate oligonucleotides (#6 and #7) were synthesized based onthe above N-terminal sequence and found to hybridize specifically to a1.1 kB EcoR1 chromosomal band from H. ducreyi strain 35000 (data notshown). Attempts to clone this fragment were unsuccessful and threeseparate vector-anchored PCR reactions (V-A PCR) were used to amplifythe relevant locus and surrounding regions (FIG. 2). Preliminarysequencing of the product of V-A PCR 1 (FIG. 2) identified an ORF thatwas homologous to the UspA2 protein of Moraxella catarrhalis and theYadA protein of Yersinia spp., but only in the C-terminal region. Sinceboth of these proteins are implicated in determining important virulencetraits (including serum resistance), additional studies were undertaken.

EXAMPLE 12 DNA and Deduced Amino Acid Sequence of the H. ducreyi dsrALocus from Strain 35000

The DNA sequence of the dsrA locus, including 100 bp of sequenceupstream of the ATG start and 126 bp of sequence downstream of the TAAtermination codon are presented in FIG. 3. The data presented wereobtained from PCR products amplified using primers 14 and 24. Sequencessimilar to −35 (TGATAA) and −10 (TATATT) E. coli promoter consensussequences were found beginning at nt 13 (TTGACA) and nt 35 (TAGAAT)respectively, and were separated by 16 nt. A putative ribosome-bindingsite (TAATGAGG) was found beginning 13 nt upstream of the dsrA startcodon. Beginning at nt 913 and ending at nt 946 was an inverted hairpinloop containing 13 matched nucleotides, consistent with a transcriptionterminator. The gene immediately downstream of dsrA and in the oppositeorientation was an ORF with homology to the hypothetical protein HI0107of the genome sequence of H. ducreyi. The GC content of the 1 kb of DNAsequence presented was 34.5%, consistent with the AT-rich nature ofHaemophilus spp. DNA.

The dsrA ORF predicted a protein of 28215 daltons, which when processedwould yield a mature protein of 26375 daltons. This is in agreement withmigration in SDS-PAGE for strain 35000 (FIG. 1). Comparison of thededuced amino acid sequence of DsrA with the N-terminal amino acidsequence revealed identity in 28 of 30 amino acids. The first tworesidues of the mature protein, QQ, were unusual in their charges;however, certain versions of mature YadA begin with two charged aminoacids (see below). Just preceding the DsrA QQ residues was the unusualsignal peptidase I cleave site of TMA. Consistent with the outermembrane localization, DsrA contained a carboxyl terminal motif endingwith a phenylalanine which is found in the majority of integral outermembrane proteins (Struyve, M. et al., Carboxyl-terminal phenylalanineis essential for the correct assembly of a bacterial outer membraneprotein. J. Mol. Biol, 218, 141–148 (1991)). The mature DsrA protein waspredicted to be very basic, with a pl of 9.1 and which accounts for itspoor transfer during Western blotting (data not shown).

Alignment of the DsrA protein with similar proteins is shown in FIG. 4.DsrA was most similar to UspA2 and YadA in a region of the C-terminusand was most divergent in the N-terminus. Using the Bestfit program,DsrA was 45% similar and 40% identical to UspA2; DsrA was 47% similarand 39% identical to YadA. It should be noted that both of theseheterologous proteins are considerable larger than DsrA which mayaccount for such differences in the N-terminal domains. The C-terminusof YadA is believed to be anchored in the outer membrane and theN-terminus encodes the functional regions of the YadA protein(Rogenkamp, A. et al., Substitution of two histidine residues in YadAprotein of Yersinia enterocolitica abrogates collagen binding, celladherence and mouse virulence. Molecular Microbiology 16, 1207–19(1995); Roggenkamp, A. et al., Deletion of amino acids 29 to 81 inadhesion protein YadA of Yersinia enterocolitica serotype 0.8 results inselective abrogation of adherence to neutrophils. Injection & Immunity65, 2506–14 (1996); Tamm, A., et al., Hydrophobic domains affect thecollagen-binding specificity, and surface polymerization as well as thevirulence potential of the YadA protein of Yersinia enterocolitica.Molecular Microbiology. 10, 995–1011 (1993)).

EXAMPLE 13 Construction and Characterization of an H. Ducreyi DsrAMutant.

An isogenic mutant (FX517, Table 1) was constructed by allelicreplacement of the wild-type locus of strain 35000. Initial attempts toobtain a double crossover with a CAT cassette in the cloned gene wereunsuccessful using pUNCH 1255 (data not shown). Therefore, we used arecently described method to obtain mutants (Bozue, J. A. et al., Facileconstruction of mutations in Haemophilus ducreyi using lacZ as acounter-selectable marker. FEMS Microbiology Letters. 164:269–73(1998)). Using this procedure, several chloramphenicol resistantcointegrates were obtained. After streaking each cointegrate onto X-galchocolate plates, several mutants were obtained for each cointegrate andnone of mutants expressed dsrA (data not shown). One mutant, FX517 wasselected for further study. Outer membranes were made from the parentand mutant strain FX517 and subjected to SDS-PAGE and Coomassie stainingor SDS-PAGE and Western blotting (FIGS. 5A and 5B, respectively). DsrAis an abundant outer membrane protein in strain 3500 but is absent inthe mutant. No reactivity was obtained from FX514 using anti-DsrAantisera (FIG. 5, Panel B) or anti-OpaF (data not shown). Similar toUspA2 and YadA, DsrA had a propensity to form multimers, especially whensolubilized at the lower temperature of 37C (FIG. 5. Panel A, and datanot shown).

The structure of the mutagenized dyrA locus in FX517 was confirmed usingSouthern blotting and PCR. In Southern blots of chromosomal DNA from theparent and mutant strains the HinCII band recognized by the dsrA probeincreased in size approximately 1 kb in the mutant as compared to theparent band. Similarly, an identical blot hybridized with the CAT proberecognized only the larger HinCII band of the mutant (data not shown).PCR, of the 35000 and FX517 dsrA locus with primers flanking the CATinsertion indicated the locus was approximately 1 kb larger in themutant (data not shown). These data are consistent with an allelicreplacement event.

EXAMPLE 14 Serum Resistance Phenotype of the DsrA Mutant

The serum susceptibility of the naturally occurring dsrA mutants and therole of the related YadA and UspA2 proteins in mediating serumresistance prompted us to test FX517 for serum sensitivity. Serumkilling studies of parent strain 35000 and dsrA mutant FX517 wereperformed using 25 and 50% normal pooled serum (FIG. 6). FX517 was verysusceptible to NHS and demonstrated zero or 2% survival in 50% and 25%NHS, respectively. In contrast, parent strain 35000 was relatively serumresistant, exhibiting 79% and 50% survival in 50% and 25% NHS (p values0.002 and 0.004 for 50% and 25% NHS, respectively, using Students pairedT test). Thus DsrA appeared to be required for expression of a serumresistant phenotype.

EXAMPLE 15 Complementation of DsrA Mutants

It was possible that a cryptic mutation had occurred during theconstruction of FX517 which could account for its serum susceptibilityphenotype. Furthermore, we wished to determine whether the serumsusceptibility of the three naturally occurring dsrA mutants could beconverted to serum resistance if they expressed dsrA. Each dsrA mutant(isogenic mutant FX517 or naturally occurring mutants CIP A75, CIP A77,and CIP 542 (Can)) was electroporated with pUNCH 1260 (dsrA) or pLSKS(vector control) plasmids. These shuttle plasmids are able to replicatein H. ducreyi. Strains containing pUNCH 1260, but not pLSKS, expresseddsrA (FIG. 7A). Subjectively, it appeared that more DsrA was expressedfrom the strains complemented with the dsrA plasmid than from 35000(n=4), perhaps due to gene dosage or growth on medium containingstreptomycin. Expression of dsrA from plasmid pUNCH 1260 suggested thatthe tentatively identified promoter (FIG. 3), was driving expression ofthe cloned dsrA gene since very little additional upstream DNA waspresent and the insert was in the opposite direction of the lac promoterin pLSKS.

Bactericidal killing was performed on each of the complemented dsrAmutants (FIG. 7B). For strains FX517, CIP A75, CIP A77, and CIP 542(Can), expression of dsrA from pUNCH 1260 conferred serum resistance.However, for strains harboring the plasmid vector lacking an insertserum resistance was not conferred.

EXAMPLE 16 Lipooligosaccharide Expression by H. Ducreyi

In some bacterial systems, mutants in LOS are more serum susceptible.Indeed, it was reported by Odumeru that the reason for the serumsusceptibility of H. ducreyi strains CIP A75 and CIP A77 was due to LOStruncation. It was possible that the lack of dsrA expression in dsrAmutants (FX517, CIP A75, CIP A77 and CIP 542 (CAN)) resulted in thetruncation of LOS directly or indirectly. Alternatively repair of dsrAexpression in LOS/dsrA apparent double mutants (CIP A75 and CIP A77)might affect LOS expression and subsequent serum susceptiblity. Toaddress these possibilities, LOS was analyzed by SDS-PAGE and silverstaining (FIG. 8) and Western blotting (data not shown). We compared35000 and FX517 LOS (without plasmids) in several silver stained gelsand the migration patterns were always indistinguishable. Furthermore.Western blotting of 35000 and FX517 LOS with anti-LOS Mab 3F11 wassimilar.

Silver stained LOS gels of the complemented dsrA mutants wereindistinguishable between each strain pair containing either pUNCH 1260(dsrA) or pLSKS, respectively. There was a minor variation in a fastermigrating LOS band for some of the strains (CIP542, no plasmid present)when grown on antibiotic free chocolate (Mueller Hinton base) ascompared to the same strain (CIP542, either plasmid present) grown onstreptomycin chocolate (Gonococcal medium base). However, it should benoted that within each pair of matched strains (expressing or notexpressing dsrA), there were no apparent major LOS differences. Thus,under the limited conditions examined here, the presence of DsrA and notLOS length was the dominant determinant of serum resistance.

EXAMPLE 17 Structural Analysis of DsrA in Other H. Ducreyi Strains

Western blotting of a variety H. ducreyi strains (FIG. 1) suggestedstrongly that DsrA varied in molecular weight and/or amino acid sequenceamong the strains. Furthermore, we desired to understand whethermutations had occurred in the naturally occurring dsrA mutants orwhether the possibility of phase variation could account for theirinability to express dsrA. PCR was used to amplify a 1.2 kb fragmentfrom 8 additional strains, including the dsrA mutants (FIG. 2, primers14 and 24). The deduced amino acid sequence indicated that overall theDsrA protein was quite similar between strains (FIG. 9; see also FIG.10). Two regions with modest variability were observed and termedvariable region 1 and 2 (VR1 and VR2). Variable region 1 included aminoacids roughly 90–100 (depending on the strain) and a few substitutionsand insertions were noted. Variable region 2 contained either 1, 2, or 3identical copies of the heptamer repeat sequence NTHNINK (SEQ ID NO:19)and spanned amino acids 174–195 in the various strains. It is likelythat the different number of repeat sequences was the predominant factoraccounting for the variable migration seen in SDS-PAGE and Westernblotting. Excepting for mutant strain CIP542(Can), which contained astop codon (see below), the sequences for all other 8 DsrA proteins wereidentical after VR2. Thus, DsrA is highly conserved in sequence, despiteits variable mobility in gels.

EXAMPLE 18 Affinity Purification of DsrA Using Vitronectin (Vn)

H. ducreyi were surface iodinated using Iodogen-coated tubes as directedby the manufacturer. Briefly, to a tube coated with 50 μg of iodogen wasadded 0.5 mCi of NaI (Amersham IMS30) and 0.5 ml of 1×109. H. ducreyi inPhosphate buffered saline (PBS). After incubation for 2 minutes, thelabeled bacteria were centrifuged and washed in medium to removeunincorporated iodine. The procedure labels primarily tyrosine residueson surfaced exposed proteins (outer membrane proteins). Each indicatedbiotinylated Vn was mixed with an aliquot of strain 35000 and strainFX517 whole surface-iodinated H. ducreyi. After Vn binding to H. ducreyistrains (15 min), unbound Vn was removed by centrifugation and washing.Bacteria and Vn were solubilized in the detergent ZW3,14 and insolublematerial removed by centrifugation for 5 min at 15,000×g. The detergentsoluble proteins were mixed with strepavidin-agarose solid phase. Afterincubation (2 hours), extensive washing the strepavidin-agarose withZW3,14 in PBS, the samples were boiled in Laemmli sample buffer, andsubjected to SDS-PAGE and autoradiography. The results are shown in FIG.12, showing that affinity purification of DsrA from whole cells ispossible using biotinylated vitronectins (Vn).

EXAMPLE 19 Method of Attachment of H. Ducreyi to Human Cells.

Efficient attachment of H. ducreyi to a keratinocyte cell line requiresDsrA expression. H. ducreyi were added to the HaCaT cells at a MOI of10:1 and incubated for 4 hours. After removal of unbound bacteria byextensive washing, CFUs were determined by plating the disruptedmonolayer. Results are shown in FIG. 11, illustrating showing thatefficient attachment of H. ducreyi to a keratinocyte cell line requiresDsrA expression. Data are from 4 experiments.

The foregoing is illustrative of the present invention and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1. An isolated polynucleotide encoding a full length Ducreyi SerumResistance A protein (DsrA), the polynucleotide selected from the groupconsisting of: (a) DNA having the nucleotide sequence of SEQ ID NO: 1;(b) a polynucleotide that hybridizes to the DNA of (a) above understringent conditions by a wash stringency of 50% Formamide with 5×Denhardt's solution, 0.5% SDS and 1× SSPE at 42° C. and which encodes afull length DrsA; and (c) a polynucleotide that differs from the DNA of(a) above due to the degeneracy of the genetic code and that encodes afull length DsrA.
 2. An isolated polynucleotide that encodes DsrA,wherein the DsrA has the amino acid sequence of SEQ ID NO:2.
 3. Theisolated polynucleotide according to claim 1 which is the DNA having thenucleotide sequence of SEQ ID NO:1.
 4. An expression vector comprisingthe polynucleotide according to claim
 1. 5. An isolated cell containingthe expression vector of claim
 4. 6. A method for detecting apolynucleotide which encodes DsrA in a biological sample, comprising:(a) contacting the complete complement of the polynucleotide sequence ofclaim 1 with the biological sample, thereby forming a hybridizationcomplex; and (b) detecting the hybridization complex, whereby thepresence of the hybridization complex detects the presence of thepolynucleotide which encodes the DsrA in the biological sample.
 7. Acomposition comprising the polynucleotide of claim 1 in apharmaceutically acceptable carrier.
 8. The composition according toclaim 7 wherein the polynucleotide is the DNA having the nucleotidesequence of SEQ ID NO:
 1. 9. A composition comprising the expressionvector of claim 4 in a pharmaceutically acceptable carrier.